Alcoholism is a widespread public health concern with limited therapeutic interventions available to help individuals battle relapse, moderate their use, and sustain abstinence. While many variables contribute to relapse vulnerability, stress is considered to play a prominent role in triggering relapse. The neuropeptide oxytocin (OT) has recently been implicated in a number of neuropsychiatric disorders, including drug and alcohol use disorders. Our lab has recently demonstrated that systemic administration of OT decreased alcohol consumption in mouse models of binge drinking and operant oral self-administration. Given that OT is known to exert anxiolytic and anti-stress effects in humans and rodents, the main overall hypothesis of this proposal is that OT (and OT receptor signaling) will be effective in attenuating stress-induced reinstatement of alcohol seeking behavior. In support of this general hypothesis, our recent preliminary data indicate that exposure to a predator odor (TMT) provoked alcohol relapse-like behavior, and this stress-induced reinstatement of alcohol seeking was attenuated by OT treatment. This research project is focused on expanding these preliminary findings to elucidate a role for the OT/OTR system in stress (TMT)-induced alcohol relapse-like behavior. Proposed studies will employ both pharmacological and chemogenetic approaches to investigate mechanisms and circuitry underlying the ability of OT to reduce stress-induced reinstatement of alcohol seeking behavior in male and female mice. Studies in Aim 1, will involve using our model of stress (TMT)-induced reinstatement of alcohol seeking to establish OT dose-effect functions in male and female mice and assess possible sex-related differences in sensitivity to stress-induced reinstatement and systemic OT treatment. Studies in Aim 2, will use designer receptor technology (viral injection of Cre-dependent DREADDs) along with Oxt-IRES-Cre transgenic mice in the same stress-induced reinstatement paradigm to (1) determine whether activation of hypothalamic oxytocin- containing neurons (via excitatory DREADDs) will mimic the effects of exogenous OT administration on stress-induced reinstatement of alcohol seeking behavior (2) assess whether selective activation of OT PVN-BNST pathway will reduce stress-induced reinstatement of alcohol seeking behavior in male and female mice and (3) determine whether blockade of oxytocin receptor signaling in the BNST will prevent the ability of chemogenetic activation of central oxytocin to attenuate stress (TMT)- induced alcohol relapse-like behavior. Results from this project will advance our understanding of neural mechanisms underlying the ability of stress to potentiate relapse behavior, as well as characterize the potential for the OT system to serve as a therapeutic target for the treatment of alcohol use disorders.